I. Field of the Invention
The present invention relates to a monoclonal antibody or fragment thereof that can recognize the aberrant form of the Cdk5 activator, p25 but not p35, which is the physiologically active form of the activator. It further relates to the hybridoma cell line, which produces the monoclonal antibody and a method of producing the antibody or fragment thereof as a recombinant protein. The invention also relates to methods for the detection and isolation of p25 or homologs thereof from biological specimens. Furthermore, the invention relates to the use of the monoclonal antibody or fragment thereof for the detection and treatment of neuronal disorders and cancer.
II. Description of Related Art
Cognitive impairment due to dementia constitutes a major symptom in patients suffering from neurodegenerative diseases such as Alzheimer's disease, vascular dementia, mixed dementia, and Parkinson's disease. Alzheimer's disease, which accounts for approximately half of all dementia cases, afflicts over 5.1 million Americans. Without additional therapeutics, it is estimated that by 2050, 11 to 16 million patients will suffer from Alzheimer's disease. Major neuropsychiatric disorders including attention deficit hyperactivity disorder (ADHD) and post-traumatic stress disorder (PTSD) also involve disorders in cognition and aversive memory.
Aberrant Cdk5 activity, resulting from the cleavage of p35 into p25, has been implicated in Alzheimer's disease (AD), tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, Huntington's disease, and Niemann-Pick disease. Cdk5 in this form associates with and contributes to the formation of neurofibrillary tangles (NFT) associated with AD brain neuropathology. Cdk5 phosphorylates the neurofilament-associated protein, tau as well as other cytoskeletal components. Dysregulation of Cdk5 results in hyperphosphorylation of tau and Cdk5 has also been found to phosphorylate the Alzheimer's precursor protein (APP). Overexpression of p25 in the cortex and hippocampus results in increased phospho-tau levels, NFT formation, and neurodegeneration. Aberrant Cdk5 activity has also been associated with hippocampal sclerosis and epilepsy, retinal degeneration, pain, and spinal chord injury.
Calpain-dependent p25 generation in response to hypoxia, ischemia, and stroke, contributes to the resulting neuronal cell loss, and Cdk5 inhibitors have been shown to mitigate cerebral tissue damage induced by stroke. Induction of aberrant Cdk5 activity has been shown to be the mode of action of numerous neurotoxic agents. Calpain cleavage of p35 occurs in response to neurotoxic insult and contributes to the neuronal cell death induced by MPTP, Aβ, maitotoxin, MPTP, organophosphates, and very low levels of methylmercury. Dysregulation of Cdk5 may also mediate the deleterious effects of the neurotoxins methamphetamine, 6-hydroxydopamine, quinolinic acid and kainic acid. Because aberrant Cdk5 activity has been so widely implicated in neurotoxicity and neurological and neurodegenerative disorders, it has been recognized as a therapeutic target for neurodegenerative disorders. However, the ability to examine the role of Cdk5 in these diseases has been hampered by the absence of a reagent that can selectively identify the Cdk5/p25 complex. Indeed, until now, the difference in the normal Cdk5/p35 complex and the disease causing Cdk5/p25 complex has been viewed as too subtle to permit differentiation of the two.
Interestingly, in a recent study it was found that overexpression of p25 in the motor control and reward center of the brain, the striatum did not result in immediate neuronal cell death but caused loss of synaptic circuitry (dendritic spines) neuroinflammation, comprised dopamine-mediated signal transduction, and impairments of dopamine-mediated behavior and motor learning (Meyer et al., 2008). Thus, neurological and neuropsychiatric disorders that may not be solely limited to neurodegeneration may arise from p25 generation. It should be noted that while the neurotoxicity and harmful effects of p25 are very well demonstrated, it can not be ruled out that the generation of low levels of p25 during strong excitotoxic excitatory glutamatergic neurotransmission may have a physiological function. One possibility is that it may recruit mitochondria in the synapse although this is only speculation at this point. Furthermore, overexpression of p25 in the parafollicular cells of the thyroid resulted in aggressive, 100% lethal medullary thyroid carcinoma, suggesting that Cdk5 and p35 or p25 are oncogenic and may be important contributors to neuroendocrine and other forms of cancer.
For research, diagnostic and pharmaceutical studies, there is a need for antibodies that selectively recognize p25, but not p35. To date, there have been no reports of such an antibody. Commercially available polyclonal antibodies to p35 also recognize p25, while there are no commercially available monoclonal antibodies to either p35 or p25. Polyclonal antibodies, although widely used, have the disadvantage in that they are of limited availability and can no longer be reproduced after the initial production. Additionally, polyclonal antibodies are a mixture of antibodies, which recognize different antigenic epitopes, and therefore it is not possible to reproduce polyclonal antibodies that recognize the same antigenic structures. Therefore, there is a need for monoclonal antibodies, which selectively and reproducibly detect p25 and not p35 thereby allowing the identification of aberrant Cdk5 activity associated with neurodegenerative diseases and cancers.